HIV reverse transcriptase (RT) is required for HIV replication and remains a primary target for anti-HIV therapies. However, due to the rapid development of HIV resistance to anti-HIV drugs, it is necessary to develop new strategies to combat HIV infection. Nucleic acid aptamers are ssDNA or RNA molecules that bind selectively and tightly to specific molecular targets. Anti-RT aptamers have been demonstrated to significantly inhibit HIV RT in enzymatic assays. Some aptamers were shown to inhibit a diverse panel of HIV RTs (universalist) while others inhibited only specific RTs (specialists). Although a few aptamers have been demonstrated to inhibit HIV replication, the bioactivity of nucleic acid aptamers has not been thoroughly explored and the potential mechanisms for the generation of HIV resistance to aptamers have not been determined. The goals of this proposal are to define the bioactivity of DNA and RNA aptamers using both single-cycle HIV replication and replication-competent HIV assays. These experiments will compare universalists and specialists to define aptamer components promoting the most potent inhibition. In addition, the project will explore the potential of HIV resistance to DNA and RNA aptamers and determine the scope of resistance generated. In particular, the type of resistance generated from universalist versus specialist aptamers will be explored. Since universalist aptamers inhibit such a diverse number of RTs, it is hoped that it will be difficult for the virus to develop resistance to these aptamers and that resistance mutations will decrease the fitness of the virus. This proposal sets a firm background for future studies to explore aptamer delivery methods more applicable to a clinical setting and also to study aptamer effects on HIV replication in the context of more complex experimental models, such as a 3D cell culture system or an animal model to examine the role of the immune system in aptamer therapeutics. Relevance to Public Health: Approximately 10% of new HIV infections are drug resistant. DNA aptamers are a possible weapon against HIV and are highly stable and easily synthesized cheaply and efficiently using technology available worldwide, but delivery methods must be modified for clinical use. RNA aptamers are also attractive candidates for therapeutics and have several advantages in delivery over DNA aptamers but have not been studied sufficiently to identify the universal components needed for successful therapy. This project will investigate both aptamers in an effort to eventually couple the advantages into one therapeutic system. In addition to the potential for therapeutic application, the project will increase understanding of HIV resistance.